Isolation of a cDNA encoding a novel member of the transglutaminase gene family from human keratinocytes. Detection and identification of transglutaminase gene products based on reverse transcription-polymerase chain reaction with degenerate primers.
We developed a method using a single set of degenerate oligonucleotide primers for amplification of the conserved active site of transglutaminases by reverse transcription-polymerase chain reaction (RT-PCR) and identification of the PCR products by cleavage with diagnostic restriction enzymes. We demonstrate amplification of tissue transglutaminase (TGC), keratinocyte transglutaminase (TGK), prostate transglutaminase (TGP), the a-subunit of factor XIII, and band 4.2 protein from different human cells or tissues. Analysis of normal human keratinocytes revealed expression of a transglutaminase different from the expected and characterized transglutaminase gene products. A full-length cDNA for the novel transglutaminase (TGX) was obtained by anchored PCR. The deduced amino acid sequence encoded a protein with 720 amino acids and a molecular mass of approximately 81 kDa. A comparison of TGX to the other members of the gene family revealed that the domain structure and the residues required for enzymatic activity and Ca2+ binding are conserved and showed an overall sequence identity of about 35%. Two transcripts with an apparent size of 2.2 and 2.8 kilobases were detected with a specific probe for TGX on Northern blots of human foreskin keratinocyte mRNA, indicating the presence of alternatively spliced mRNAs. cDNA sequencing revealed a shorter TGX transcript lacking the sequence homologous to that encoded by exon III of other transglutaminase genes. TGX expression increased severalfold when keratinocyte cultures were induced to differentiate by suspension or growth to postconfluency, suggesting that TGX contributes to the formation of the cornified envelope.
Aeschlimann
D
D
Division of Orthopedic Surgery, University of Wisconsin, Madison, Wisconsin 53792, USA.
Koeller
M K
MK
Allen-Hoffmann
B L
BL
Mosher
D F
DF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D006801:Humans; D015603:Keratinocytes / Q000201:enzymology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D012326:RNA Splicing; D017386:Sequence Homology, Amino Acid; D011503:Transglutaminases / Q000737:chemistry / Q000235:genetics*; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D018076:DNA, Complementary; D011503:Transglutaminases